Simultaneous membrane interaction of amphipathic peptide monomers, self-aggregates and cargo complexes detected by fluorescence correlation spectroscopy

摘要

Abstract Peptides able to translocate cell membranes while carrying macromolecular cargo, as cell-penetrating peptides (CPPs), can contribute to the field of drug delivery by enabling the transport of otherwise membrane impermeable molecules. Formation of non-covalent complexes between amphipathic peptides and oligonucleotides is driven by electrostatic and hydrophobic interactions. Here we investigate and quantify the coexistence of distinct molecular species in multiple equilibria, namely peptide monomer, peptide self-aggregates and peptide/oligonucleotide complexes. As a model for the complexes, we used a stearylated peptide from the PepFect family, PF14 and siRNA. PF14 has a cationic part and a lipid part, resembling some characteristics of cationic lipids. Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) were used to detect distinct molecular entities in solution and at the plasma membrane of live cells. For that, we labeled the peptide with carboxyrhodamine 6G and the siRNA with Cyanine 5. We were able to detect fluorescent entities with diffusional properties characteristic of the peptide monomer as well as of peptide aggregates and peptide/oligonucleotide complexes. Strategies to avoid peptide adsorption to solid surfaces and self-aggregation were developed and allowed successful FCS measurements in solution and at the plasma membrane. The ratio between the detected molecular species was found to vary with pH, peptide concentration and the proximity to the plasma membrane. The present results suggest that the diverse cellular uptake mechanisms, often reported for amphipathic CPPs, might result from the synergistic effect of peptide monomers, self-aggregates and cargo complexes, distributed unevenly at the plasma membrane. Graphical abstract Display Omitted Highlights ? The amphipathic, N-stearylated CPP, PF14 forms aggregates with heterogeneous shape and polydisperse size. ? The ratio of peptide monomers to self-aggregates was successfully determined using FCS. ? The ratio of peptide monomers to self-aggregates varies with pH, peptide concentration and proximity to the plasma membrane. ? Peptide self-aggregates and non-covalent complexes with siRNA are dynamic structures in equilibria. ? Peptide monomers, self-aggregates and PF14/siRNA complexes were detected simultaneously at the plasma membrane.